Novel g-protein coupled receptors and dna sequences thereof

ABSTRACT

The present invention relates to in particular immune modulatory polypeptides and polynucleotides, recombinant materials and methods for their production. Such polypeptides and polynucleotides are of interest in relation to methods of treatment of diseases whereby immune responses initiated by dendritic cells (DC), monocytes or lymphocytes, play a causal or contributory role.

FIELD OF THE INVENTION

This invention relates to newly identified polypeptides andpolynucleotides encoding such polypeptides, to their use in diagnosisand in identifying compounds that may be agonists, antagonists that arepotentially useful in therapy, and to production of such polypeptidesand polynucleotides, belonging to the class of G-protein coupledreceptors.

BACKGROUND OF THE INVENTION

The drug discovery process is currently undergoing a fundamentalrevolution as it embraces “functional genomics”, that Is, highthroughput genome- or gene-based biology. This approach as a means toidentify genes and gene products as therapeutic targets is rapidlysuperceding earlier approaches based on “positional cloning”. Aphenotype, that is a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

Functional genomics relies heavily on high-throughput DNA sequencingtechnologies and the various tools of bioinformatics to Identify genesequences of potential interest from the many molecular biologydatabases now available. There is a continuing need to identify andcharacterise further genes and their related polypeptides/proteins, astargets for drug discovery.

SUMMARY OF THE INVENTION

The present invention relates to in particular immune modulatorypolypeptides and polynucleotides, recombinant materials and methods fortheir production. Such polypeptides and polynucleotides are of interestin relation to methods of treatment of diseases whereby immune responsesinitiated by dendritic cells (DC), monocytes or lymphocytes, play acausal or contributory role. Such diseases include but are not limitedto chronic inflammatory diseases such as inflammatory bowel disease,autoimmune diseases such as rheumatoid arthritis or systemic lupuserytomatosis or multiple sclerosis, transplant rejection crisis,inflammatory skin diseases such as contact hypersensitivity or atopicdermatitis; and diseases or syndromes in which a significantpathological component is immune suppression as in, and including, AIDSand cancer.

Hereafter referred to as “diseases of the invention”. In a furtheraspect, the Invention relates to methods for identifying agonists andantagonists (e.g.inhibitors) using the materials provided by theinvention, and treating conditions associated with monocyte/macrophageand DC or lymphocyte migration/activation, regulation of immune celldifferentiation, cytokine production, release of inflammatory mediators,regulation of inflammation, modulation of immune responses with theidentified compounds. In a still further aspect, the invention relatesto diagnostic assays for detecting diseases associated withinappropriate activity/levels of monocyte/macrophage, DC and lymphocytecell migration/activation, leading to chronic inflammatory conditions,skin hypersensitivity reactions, self-destructive immune responses, andimmune-suppressed states.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to immune modulatorypolypeptides. Such polypeptides include:

-   -   (a) an isolated polypeptide encoded by a polynucleotide        comprising the sequence of SEQ ID NO 1 or SEQ ID NO 3    -   (b) an isolated polypeptide comprising a polypeptide sequence        having at least 95%, 96%, 97%, 98%, or 99% identity to the        polypeptide sequence of SEQ ID NO 2 or SEQ ID NO 4;    -   (c) an isolated polypeptide comprising the polypeptide sequence        of SEQ ID NO NO 2 or SEQ ID NO 4;    -   (d) an isolated polypeptide having at least 95%, 96%, 97%, 98%,        or 99% identity to the polypeptide sequence of SEQ ID NO 2 or        SEQ ID NO 4;    -   (e) the polypeptide sequence of SEQ ID NO 2 or SEQ ID NO 4; and    -   (f) an isolated polypeptlde having or comprising a polypeptide        sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98,        or 0.99 compared to the polypeptide sequence of SEQ ID NO 2 or        SEQ ID NO 4;    -   (g) fragments and variants of such polypeptides in (a) to (f).

Polypeptides of the present invention are believed to be members of theG protein-coupled receptors family of polypeptides. The biologicalproperties of the immune modulatory polypeptides are hereinafterreferred to as “biological activity of immune modulators” or “immunemodulatory activity” including monocyte/macrophage migration/activation,regulation of dendritic cell differentiation, regulation of lymphocyteactivation, proliferation and differentiation regulation ofinflammation, regulation of cytokine production and/or release,regulation of pro-inflammatory mediator production and/or release.Polypeptides of the present invention also includes variants of theaforementioned polypeptides, including all allelic forms and splicevariants. Such polypeptides vary from the reference polypeptide byInsertions, deletions, and substitutions that may be conservative ornon-conservative, or any combination thereof. Particularly preferredvariants are those in which several, for instance from 50 to 30, from 30to 20, from 20 to 10, from to 5, from 5 to 3, from 3 to 2, from 2 to 1or 1 amino acids are inserted, substituted, or deleted, in anycombination.

Preferred fragments of polypeptides of the present invention include anisolated polypeptide comprising an amino acid sequence having at least30, 50 or 100 contiguous amino acids from the amino acid sequence of SEQID NO 2 or SEQ ID NO. 4 or an isolated polypeptide comprising an aminoacid sequence having at least 30, 50 or 100 contiguous amino acidstruncated or deleted from the amino acid sequence of SEQ ID NO 2 or SEQID NO. 4 Preferred fragments are biologically active fragments thatmediate the biological activity of monocyte/macrophagemigration/activation, the immune modulatory polypeptides are hereinafterreferred to as “biological activity of immune modulators” or “immunemodulatory activity” including monocyte/macrophage migration/activation,regulation of dendritic cell differentiation, regulation of lymphocyteactivation, proliferation and differentiation, regulation ofinflammation, regulation of cytokine production and/or release,regulation of pro-inflammatory mediator production and/or releaseincluding those with a similar activity or an improved activity, or witha decreased undesirable activity. Also preferred are those fragmentsthat are antigenic or immunogenic in an animal, especially in a human.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention. Thepolypeptides of the present invention may be in the form of the “mature”protein or maybe a part of a larger protein such as a precursor or afusion protein. It is often advantageous to include an additional aminoacid sequence that contains secretory or leader sequences,pro-sequences, sequences that aid in purification, for instance multiplehistidine residues, or an additional sequence for stability duringrecombinant production.

Polypeptides of the present invention can be prepared in any suitablemanner, for instance by isolation form naturally occurring sources, fromgenetically engineered host cells comprising expression systems (videinfra) or by chemical synthesis, using for instance automated peptidesynthesizers, or a combination of such methods. The means for preparingsuch polypeptides are well understood in the art.

In a further aspect, the present invention relates to immune modulatorypolynucleotides. Such polynucleotides include:

-   -   (a) an isolated polynucleotide comprising a polynucleotide        sequence having at least 95%, 96%, 97%, 98%, or 99% identity to        the polynucleotide sequence of SEQ ID NO. 1 or SEQ ID NO. 3; (b)        an isolated polynucleotide comprising the polynucleotide of SEQ        ID NO. 1 or SEQ ID NO. 3; (c) an isolated polynucleotide having        at least 95%, 96%, 97%, 98%, or 99% identity to the        polynucleotide of SEQ ID NO 1 or SEQ ID NO. 3;    -   (d) the isolated polynucleotide of SEQ ID NO 1 or SEQ ID NO. 3;    -   (e) an isolated polynucleotide comprising a polynucleotide        sequence encoding a polypeptide sequence having at least 95%,        96%, 97%, 98%, or 99% identity to the polypeptide sequence of        SEQ ID NO 2 or SEQ ID NO.4;    -   (f) an isolated polynucleotide comprising a polynucleotide        sequence encoding the polypeptide of SEQ ID NO 2 or SEQ ID NO.4;    -   (g) an isolated polynucleotide having a polynucleotide sequence        encoding a polypeptide sequence having at least 95%, 96%, 97%,        98%, or 99% identity to the polypeptide sequence of SEQ ID NO. 2        or SEQ ID NO.4;    -   (h) an isolated polynucleotide encoding the polypeptide of SEQ        ID NO. 2 or SEQ ID NO.4;    -   (i) an isolated polynucleotide having or comprising a        polynucleotide sequence that has an Identity Index of 0.95,        0.96, 0.97, 0.98, or 0.99 compared to the polynucleotide        sequence of SEQ ID NO. 2 or SEQ ID NO.4 an isolated        polynucleotide having or comprising a polynucleotide sequence        encoding a polypeptide sequence that has an Identity Index of        0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide        sequence of SEQ ID NO 2 or SEQ ID NO.4; and polynucleotides that        are fragments and variants of the above mentioned        polynucleotides or that are complementary to above mentioned        polynucleotides, over the entire length thereof.

Preferred fragments of polynucleotides of the present invention includean isolated polynucleotide comprising an nucleotide sequence having atleast 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQID NO 1 or SEQ ID NO 3, or an isolated polynucleotide comprising ansequence having at least 30, 50 or 100 contiguous nucleotides truncatedor deleted from the sequence of SEQ ID NO 1 or SEQ ID NO 3.

Preferred variants of polynucleotides of the present invention includesplice variants, allelic variants, and polymorphisms,includingpolynucleotides having one or more single nucleotide polymorphisms(SNPs).

Polynucleotides of the present invention also include polynucleotidesencoding polypeptide variants that comprise the amino acid sequence ofSEQ ID NO 2 or SEQ ID NO 4 and in which several, for instance from 50 to30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to2, from 2 to 1 or 1 amino acid residues are substituted, deleted oradded, in any combination.

In a further aspect, the present invention provides polynucleotides thatare RNA transcripts of the DNA sequences of the present invention.Accordingly, there is provided an RNA polynucleotide that:

-   -   (a) comprises an RNA transcript of the DNA sequence encoding the        polypeptide of SEQ ID NO 2or SEQ ID NO 4;    -   (b) is the RNA transcript of the, DNA sequence encoding the        polypeptide of SEQ ID NO 2 or SEQ ID NO 4;    -   (c) comprises an RNA transcript of the DNA sequence of SEQ ID NO        1 or SEQ ID NO. 3; or    -   (d) is the RNA transcript of the DNA sequence of SEQ ID NO 1 or        SEQ ID NO 3; and RNA polynucleotides that are complementary        thereto.

The polynucleotide sequence of SEQ ID NO 1 is a cDNA sequence thatencodes the polypeptide of SEQ ID NO 2. The polynucleotide sequence ofSEQ ID NO 3 is a cDNA sequence that encodes the polypeptide of SEQ ID NO4. The polynucleotide sequence encoding the polypeptide of SEQ ID NO 2may be identical to the polypeptide encoding sequence of SEQ ID NO 1 orit may-be a sequence other than SEQ ID NO 1, which, as a result of theredundancy (degeneracy) of the genetic code, also encodes thepolypeptide of SEQ ID NO 2. The polynucleotide sequence encoding thepolypeptide of SEQ ID NO 4 may be identical to the polypeptide encodingsequence of SEQ ID NO 3 or it may-be a sequence other than SEQ ID NO 3,which, as a result of the redundancy (degeneracy) of the genetic code,also encodes the polypeptide of SEQ ID NO 4. The polypeptide of the SEQID NO 2 or SEQ ID NO 4 is related to other proteins of the Gprotein-coupled receptors family, having homology and/or structuralsimilarity with GPCR-LYMST Jensen, C. P. et al., Proc. Natl. Acad. Sci.U.S.A. 91: 4816-4820,1994).

Preferred polypeptides and polynucleotides of the present invention areexpected to have, inter alia, similar biological functions/properties totheir homologous polypeptides and polynucleotides. Furthermore,preferred polypeptides and polynucleotides of the present invention haveat least one activity such as: association with monocyte/macrophage andDC or lymphocyte migration/activation, regulation of immune celldifferentiation, cytokine production, release of inflammatory mediators,regulation of inflammation, or modulation of immune responses.

Polynucleotides of the present invention may be obtained using standardcloning and screening techniques from a cDNA library derived from mRNAin cells of human adult or fetal tissue (see for instance, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). Polynucleotides ofthe invention can also be obtained from natural sources such as genomicDNA libraries or can be synthesized using well known and commerciallyavailable techniques.

When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself, or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- orprepro-protein sequence, or other fusion peptide portions. For example,a marker sequence that facilitates purification of the fused polypeptidecan be encoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et aL, Proc NatlAcad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide mayalso contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

Polynucleotides that are identical, or have sufficient identity to apolynucleotide sequence of SEQ ID NO 1. or SEQ ID NO 3 may be used ashybridization probes for cDNA and genomic DNA or as primers for anucleic acid amplification reaction (for instance, PCR). Such probes andprimers may be used to isolate full-length cDNAs and genomic clonesencoding polypeptides of the present invention and to isolate cDNA andgenomic clones of other genes (including genes encoding paralogs fromhuman sources and orthologs and paralogs from species other than human)that have a high sequence similarity to SEQ ID NO: 1 or SEQ ID NO 3,typically at least 95% identity. Preferred probes and primers willgenerally comprise at least 15 nucleotides, preferably, at least 30nucleotides and may have at least 50, if not at least 100 nucleotides.Particularly preferred probes will have between 30 and 50 nucleotides.Particularly preferred primers will have between 20 and 25 nucleotides.

A polynucleotide encoding a polypeptide of the present invention,including homologs from species other than human, may be obtained by aprocess comprising the steps of screening a library under stringenthybridization conditions with a labeled probe having the sequence of SEQID NO: 1 or SEQ ID NO 3 or a fragment thereof, preferably of at least 15nucleotides; and isolating full-length cDNA and genomic clonescontaining said polynucleotide sequence. Such hybridization techniquesare well known to the skilled artisan. Preferred stringent hybridizationconditions include overnight incubation at 420 C in a solutioncomprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA;followed by washing the filters in 0.1×SSC at about 650 C. Thus thepresent invention also includes isolated polynucleotides, preferablywith a nucleotide sequence of at least 100, obtained by screening alibrary under stringent hybridization conditions with a labeled probehaving the sequence of SEQ ID NO: 1 or SEQ ID NO 3 or a fragmentthereof, preferably of at least 15 nucleotides.

The person skilled in the art will appreciate that, in many cases, anisolated cDNA sequence will be incomplete, in that the region coding forthe polypeptide does not extend all the way through to the 5′ terminus.This is a consequence of reverse transcriptase, an enzyme withinherently low 11 processivity” (a measure of the ability of the enzymeto remain attached to the template during the polymerisation reaction),failing to complete a DNA copy of the mRNA template during first strandcDNA synthesis.

There are several methods available and well known to those skilled Inthe art to obtain full-length cDNAs, or extend short cDNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman et al., Proc Nat Acad Sci USA 85, 8998-9002,1988). Recent modifications of the technique, exemplified by theMarathon (trade mark) technology (Clontech Laboratories Inc.) forexample, have significantly simplified the search for longer cDNAs. Inthe Marathon (trade mark) technology, cDNAs have been prepared from mRNAextracted from a chosen tissue and an ‘adaptor’ sequence ligated ontoeach end. Nucleic acid amplification (PCR) is then carried out toamplify the “missing” 5′ end of the cDNA using a combination of genespecific and adaptor specific oligonucleotide primers. The PCR reactionis then repeated using ‘nested’ primers, that is, primers designed toanneal within the amplified product (typically an adaptor specificprimer that anneals further 3′ in the adaptor sequence and a genespecific primer that anneals further 5′ in the known gene sequence). Theproducts of this reaction can then be analysed by DNA sequencing and afull-length cDNA constructed either by joining the product directly tothe existing cDNA to give a complete sequence, or carrying out aseparate full-length PCR using the new sequence information for thedesign of the 5′ primer.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in the art from genetically engineered host cellscomprising expression systems. Accordingly, in a further aspect, thepresent invention relates to expression systems comprising apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression systems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Polynucleotides may be introduced into hostcells by methods described in many standard laboratory manuals, such asDavis et al., Basic Methods in Molecular Biology (1986) and Sambrook etal.(ibid).

Preferred methods of introducing polynucleotides into host cellsinclude, for instance, calcium phosphate transfection, DEAE-dextranmediated transfection, transvection, microinjection, cationiclipid-mediated transfection, electroporation, transduction, scrapeloading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as Streptococci, Staphylococci, E coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C1 27, 3T3, BHK, HEK 293 and Bowesmelanoma cells; and plant cells.

A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector that is able to maintain, propagate or express apolynucleotide to produce a polypeptide In a host may be used. Theappropriate polynucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al (see above).Appropriate secretion signals may be incorporated into the desiredpolypeptide to allow secretion of the translated protein into the lumenof the endoplasmic reticulum, the periplasmic space or the extracellularenvironment. These signals may be endogenous to the polypeptide or theymay be heterologous signals.

If a polypeptide of the present invention is to be expressed for use inscreening assays, it is generally preferred that the polypeptide beproduced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If the polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

Polypeptides of the present invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured duringintracellular synthesis, isolation and/or purification.

Polynucleotides of the present invention may be used as diagnosticreagents, through detecting mutations in the associated gene. Detectionof a mutated form of the gene characterised by the polynucleotide of SEQID NO 1 or SEQ ID NO 3 in the cDNA or genomic sequence and which isassociated with a dysfunction will provide a diagnostic tool that canadd to, or define, a diagnosis of a disease, or susceptibility to adisease, which results from under-expression, over-expression or alteredspatial or temporal expression of the gene. Individuals carryingmutations in the gene may be detected at the DNA level by a variety oftechniques well known in the art.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or it may be amplifiedenzymatically by using PCR, preferably RT-PCR, or other amplificationtechniques prior to analysis. RNA or cDNA may also be used in similarfashion. Deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Pointmutations can be Identified by hybridizing amplified DNA to labelednucleotide sequences. Perfectly matched sequences can be distinguishedfrom mismatched duplexes by RNase digestion or by differences in meltingtemperatures.

DNA sequence difference may also be detected by alterations in theelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing (see, for instance, Myerset aL, Science (1985) 230:1242). Sequence changes at specific locationsmay also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (see Cotton et aL, ProcNatl Acad Sci USA (1985) 85: 4397-4401).

An array of oligonucleotides probes comprising immune modulatorypolynucleotide sequence or fragments thereof can be constructed toconduct efficient screening of e.g., genetic mutations. Such arrays arepreferably high density arrays or grids. Array technology methods arewell known and have general applicability and can be used to address avariety of questions in molecular genetics including gene expression,genetic linkage, and genetic variability, see, for example, M.Chee etal., Science, 274, 610-613 (1996) and other references cited therein.

Detection of abnormally decreased or increased levels of polypeptide ormRNA expression may also be used for diagnosing or determiningsusceptibility of a subject to a disease of the invention. Decreased orincreased expression can be measured at the RNA level using any of themethods well known in the art for the quantitation of polynucleotides,such as, for example, nucleic acid amplification, for instance PCR,RT-PCR, RNase protection, Northern blotting and other hybridizationmethods. Assay techniques that can be used to determine levels of aprotein, such as a polypeptide of the present invention, in a samplederived from a host are well-known to those skilled in the art. Suchassay methods include radioimmunoassays, competitive-binding assays,Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagonostickit comprising:

-   -   (a) a polynucleotide of the present invention, preferably the        nucleotide sequence of SEQ ID NO 1 or SEQ ID NO 3, or a fragment        or an RNA transcript thereof;    -   (b) a nucleotide sequence complementary to that of (a);    -   (c) a polypeptide of the present invention, preferably the        polypeptide of SEQ ID NO 2 or SEQ ID NO 4 or a fragment thereof;        or    -   (d) an antibody to a polypeptide of the present invention,        preferably to the polypeptide of SEQ ID NO 2 or SEQ ID NO 4.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, particularlydiseases of the invention, amongst others.

The polynucleotide sequences of the present invention are valuable forchromosome localisation studies. The sequence is specifically targetedto, and can hybridize with, a particular location on an Individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound in, for example, V. McKusick, Mendelian Inheritance in Man(available on-line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (co-inheritance of physically adjacent genes). Precisehuman chromosomal localisations for a genomic sequence (gene fragmentetc.) can be determined using Radiation Hybrid (RH) Mapping (Walter, M.Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P., (1994) Amethod for constructing radiation hybrid maps of whole genomes, NatureGenetics 7, 22-28). A number of RH panels are available from ResearchGenetics (Huntsville, Ala., USA) e.g. the—GeneBridge4 RH panel (Hum MoiGenet Mar. 1996;5(3):339-46 A radiation hybrid map of the human genome.Gyapay G, Schmitt K, Fizames C, Jones H, Vega-Czarny N, Spilleft D,Muselet D, Prud'Homme J F, Dib C, Auffray C, Morissette J, WeissenbachJ, Goodfellow P N). To determine the chromosomal location of a geneusing this panel, 93 PCRs are performed using primers designed from thegene of interest on RH IDNAs. Each of these DNAs contains random humangenomic fragments maintained in a hamster background (human/hamsterhybrid cell lines). These PCRs result in 93 scores indicating thepresence or absence of the PCR product of the gene of interest. Thesescores are compared with scores created using PCR products from genomicsequences of known location. This comparison is conducted athftp://www.genome.wi.mit.edu/.

The polynucleotide sequences of the present invention are also valuabletools for tissue expression studies. Such studies allow thedetermination of expression patterns of polynucleotides of the presentinvention which may give an indication as to the expression patterns ofthe encoded polypeptides in tissues, by detecting the mRNAs that encodethem. The techniques used are well known in the art and include in situhydridisation techniques to clones arrayed on a grid, such as cDNAmicroarray hybridisation (Schena et al, Science, 270, 467-470, 1995 andShalon et a], Genome Res, 6, 639-645, 1996) and nucleotide amplificationtechniques such as PCR. A preferred method uses the TAQMAN (Trade mark)technology available from Perkin Elmer. Results from these studies canprovide an indication of the normal function of the polypeptide in theorganism. In addition, comparative studies of the normal expressionpattern of mRNAs with that of mRNAs encoded by an alternative form ofthe same gene (for example, one having an alteration in polypeptidecoding potential or a regulatory mutation) can provide valuable insightsinto the role of the polypeptides of the present invention, or that ofinappropriate expression thereof in disease. Such inappropriateexpression may be of a temporal, spatial or simply quantitative nature.

The polypeptides of the present invention are expressed in tissues andtissues related to associated with monocyte/macrophage and DC orlymphocyte migration/activation, regulation of immune celldifferentiation, cytokine production, release of inflammatory mediators,regulation of inflammation, or modulation of immune responses.

A further aspect of the present invention relates to antibodies. Thepolypeptides of the invention or their fragments, or cells expressingthem, can be used as immunogens to produce antibodies that areimmunospecific for polypeptides of the present invention. The term“immunospecific” means that the antibodies have substantially greateraffinity for the polypeptides of the invention than their affinity forother related polypeptides in the prior art. Antibodies generatedagainst polypeptides of the present invention may be obtained byadministering the polypeptides or epitope-bearing fragments, or cells toan animal, preferably a non-human animal, using routine protocols. Forpreparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Mistein, C.,Nature (1975) 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et aL, Immunology Today (1983) 4:72) and theEBV-hybridoma technique (Cole et aL, Monoclonal Antibodies and CancerTherapy, 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies, such as thosedescribed in U.S. Pat. No. 4,946,778, can also be adapted to producesingle chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography. Antibodies against polypeptides of the presentinvention may also be employed to treat diseases of the invention,amongst others.

Polypeptides and polynucleotides of the present invention may also beused as vaccines. Accordingly, in a further aspect, the presentinvention relates to a method for inducing an immunological response ina mammal that comprises inoculating the mammal with a polypeptide of thepresent invention, adequate to produce antibody and/or T cell immuneresponse, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said animal from disease, whether thatdisease is already established within the individual or not. Animmunological response in a mammal may also be induced by a methodcomprises delivering a polypeptide of the present invention via a vectordirecting expression of the poiynucleotide and coding for thepolypeptide in vivo in order to induce such an immunological response toproduce antibody to protect said animal from diseases of the invention.One way of administering the vector is by accelerating it into thedesired cells as a coating on particles or otherwise. Such nucleic acidvector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNAhybrid. For use a vaccine, a polypeptide or a nucleic acid vector willbe normally provided as a vaccine formulation (composition). Theformulation may further comprise a suitable carrier. Since a polypeptidemay be broken down-in the stomach, it is preferably administeredparenterally (for instance, subcutaneous, intramuscular, intravenous, orintradermal injection). Formulations suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions that may contain anti-oxidants, buffers, bacteriostats andsolutes that render the formulation isotonic with the blood of therecipient; and aqueous and non-aqueous sterile suspensions that mayinclude suspending agents or thickening agents.

The formulations may be presented in unit-dose or multi-dose containers,for example, sealed ampoules and vials and may be stored in afreeze-dried condition requiring only the addition of the sterile liquidcarrier immediately prior to use. The vaccine formulation may alsoinclude adjuvant systems for enhancing the immunogenicity of theformulation, such as oil-in water systems and other systems known in theart. The dosage will depend on the specific activity of the vaccine andcan be readily determined by routine experimentation.

Polypeptides of the present invention have one or more biologicalfunctions that are of relevance in one or more disease states, inparticular the diseases of the invention hereinbefore mentioned. It istherefore useful to identify compounds that stimulate or inhibit thefunction or level of the polypeptide. Accordingly, in a further aspect,the present invention provides for a method of screening compounds toidentify those that stimulate or inhibit the function or level of thepolypeptide. Such methods identify agonists or antagonists that may beemployed for therapeutic and prophylactic purposes for such diseases ofthe invention as hereinbefore mentioned. Compounds may be identifiedfrom a variety of sources, for example, cells, cell-free preparations,chemical libraries, collections of chemical compounds, and naturalproduct mixtures. Such agonists or antagonists so-identified may benatural or modified substrates, ligands, receptors, enzymes, etc., asthe case may be, of the polypeptide; a structural or functional mimeticthereof (see Coligan et aL, Current Protocols in Immunology 1(2):Chapter5 (1991)) or a small molecule.

The screening method may simply measure the binding of a candidatecompound to the polypeptide, or to cells or membranes bearing thepolypeptide, or a fusion protein thereof, by means of a label directlyor indirectly associated with the candidate compound. Alternatively, thescreening method may involve measuring or detecting (qualitatively orquantitatively) the competitive binding of a candidate compound to thepolypeptide against a labeled competitor (e.g. agonist or antagonist).Further, these screening methods may test whether the candidate compoundresults in a signal generated by activation or inhibition of thepolypeptide, using detection systems appropriate to the cells bearingthe polypeptide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution containing a polypeptide of the presentinvention, to form a mixture, measuring a HGRL101 activity in themixture, and comparing the HGRL101 activity of the mixture to a controlmixture which contains no candidate compound.

Polypeptides of the present invention may be employed in conventionallow capacity screening methods and also in high-throughput screening(HTS) formats. Such HTS formats include not only the well-establisheduse of 96- and, more recently, 384-well micotiter plates but alsoemerging methods such as the nanowell method described by Schullek etal, Anal Biochem., 246, 20-29, (1997).

Fusion proteins, such as those made from Fc portion and immunemodulatory polypeptide, as hereinbefore described, can also be used forhigh-throughput screening assays to identify antagonists for thepolypeptide of the present invention (see D. Bennett et aL, J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

Screening Techniques

The polynucleotides, polypeptides and antibodies to the polypeptide ofthe present invention may also be used to configure screening methodsfor detecting the effect of added compounds on the production of mRNAand polypeptide in cells. For example, an ELISA assay may be constructedfor measuring secreted or cell associated levels of polypeptide usingmonoclonal and polyclonal antibodies by standard methods known in theart. This can be used to discover agents that may inhibit or enhance theproduction of polypeptide (also called antagonist or agonist,respectively) from suitably manipulated cells or tissues.

A polypeptide of the present invention may be used to identify membranebound or soluble receptors, if any, through standard receptor bindingtechniques known in the art. These include, but are not limited to,ligand binding and crosslinking assays in which the polypeptide islabeled with a radioactive isotope (for instance, 1251), chemicallymodified (for instance, biotinylated), or fused to a peptide sequencesuitable for detection or purification, and incubated with a source ofthe putative receptor (cells, cell membranes, cell supernatants, tissueextracts, bodily fluids). Other methods include biophysical techniquessuch as surface plasmon resonance and spectroscopy. These screeningmethods may also be used to identify agonists and antagonists of thepolypeptide that compete with the binding of the polypeptide to itsreceptors, if any. Standard methods for conducting such assays are wellunderstood in the art.

Examples of antagonists of polypeptides of the present invention includeantibodies or, in some cases, oligonucleotides or proteins that areclosely related to the ligands, substrates, receptors, enzymes, etc., asthe case may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or a small molecule that bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented.

Screening methods may also involve the use of transgenic technology anda immune modulatory gene. The art of constructing transgenic animals iswell established. For example, the immune modulatory gene may beintroduced through microinjection into the male pronucleus of fertilizedoocytes, retroviral transfer into pre- or post-implantation embryos, orinjection of genetically modified, such as by electroporation, embryonicstem cells into host blastocysts. Particularly useful transgenic animalsare so-called “knock-in” animals in which an animal gene Is replaced bythe human equivalent within the genome of that animal. Knock-Intransgenic animals are useful in the drug discovery process, for targetvalidation, where the compound is specific for the human target. Otheruseful transgenic animals are so-called “knock-out” animals in which theexpression of the animal ortholog of a polypeptide of the presentinvention and encoded by an endogenous DNA sequence in a cell ispartially or completely annulled. The gene knock-out may be targeted tospecific cells or tissues, may occur only in certain cells or tissues asa consequence of the limitations of the technology, or may occur in all,or substantially all, cells in the animal. Transgenic animal technologyalso offers a whole animal expression-cloning system in which introducedgenes are expressed to give large amounts of polypeptides of the presentinvention.

Screening kits for use in the above described methods form a furtheraspect of the present invention. Such screening kits comprise:

-   -   (a) a polypeptide of the present invention;    -   (b) a recombinant cell expressing a polypeptide of the present        invention,    -   (c) a cell membrane expressing a polypeptide of the present        invention; or    -   (d) an antibody to a polypeptide of the present invention; which        polypeptide is preferably that of SEQ ID NO 2 or SEQ ID. No.4.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

Glossary

The following definitions are provided to facilitate understanding ofcertain terms used frequently hereinbefore.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

“Isolated” means altered by the human hands from its natural state, ie.if it occurs in nature, it has been changed or removed from its originalenvironment, or both. For example, a polynucleotide or a polypeptidenaturally present in a living organism is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated”, as the term is employed herein.Moreover, a polynucleotide or polypeptide that is introduced into anorganism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Polynucleotide” generally refers to any polyribonucleotide (RNA) orpolydeoxribonucleotide (DNA), which may be unmodified or modified RNA orDNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons.

“Modified” bases include, for example, tritylated bases and unusualbases such as inosine. A variety of modifications may be made to DNA andRNA; thus, “polynucleotide” embraces chemically, enzymatically ormetabolically modified forms of polynucleotides as typically found innature, as well as the chemical forms of DNA and RNA characteristic ofviruses and cells. “Polynucleotide” also embraces relatively shortpolynucleotides, often referred to as oligonucleotides.

“Polypeptide” refers to any polypeptide comprising two or more aminoacids joined to each other by peptide bonds or modified peptide bonds,—i.e., peptide isosteres. “Polypeptide” refers to both short chains,commonly referred to as peptides, oligopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, Including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.

It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, biotinylation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutarnate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racernization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination (see, for instance,Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York, 1993; Wold, F., Post-translationalProtein Modifications: Perspectives and Prospects, 1-12, InPost-translational Covalent Modification of Proteins, B. C. Johnson,Ed., Academic Press, New York, 1983; Seifter et aL, “Analysis forprotein modifications and nonprotein cofactors”, Meth Enzymol, 182,626-646, 1990, and Rattan et al., “Protein Synthesis: Post-translationalModifications and Aging”, Ann NY Acad Sci, 663, 48-62, 1992).

“Fragment” of a polypeptide sequence refers to a polypeptide sequencethat is shorter than the reference sequence but that retains essentiallythe same biological function or activity as the reference polypeptide.“Fragment” of a polynucleotide sequence refers to a polynucloetidesequence that is shorter than the reference sequence of SEQ ID NO 1 orSEQ ID NO 3.

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains the essentialproperties thereof. A typical variant of a polynucleotide differs innucleotide sequence from the reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from the referencepolypeptide. Generally, alterations are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, insertions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. Typicalconservative substitutions include Gly, Ala; Val, lie, Leu; Asp, Glu;Asn, Gln-I Ser, Thr; Lys, Arg; and Phe and Tyr. A variant of apolynucleotide or polypeptide may be naturally occurring such as anallele, or it may be a variant that is not known to occur naturally.Non-naturally occurring variants of polynucleotides and polypeptides maybe made by mutagenesis techniques or by direct synthesis. Also includedas variants are polypeptides having one or more post-translationalmodifications, for instance glycosylation, phosphorylation, methylation,ADIP ribosylation and the like. Embodiments include methylation of theN-terminal amino acid, phosphorylations of serines and threonines andmodification of C-terminal glycines.

“Allele” refers to one of two or more alternative forms of a geneoccuring at a given locus in the genome.

“Polymorphism” refers to a variation in nucleotide sequence (and encodedpolypeptide sequence, if relevant) at a given position in the genomewithin a population.

“Single Nucleotide Polymorphism” (SNP) refers to the occurence ofnucleotide variability at a single nucleotide position in the genome,within a population. An SNP may occur within a gene or within intergenicregions of the genome. SNPs can be assayed using Allele SpecificAmplification (ASA). For the process at least 3 primers are required. Acommon primer is used in reverse complement to the polymorphism beingassayed. This common primer can be between 50 and 1500 bps from thepolymorphic base. The other two (or more) primers are identical to eachother except that the final 3′ base wobbles to match one of the two (ormore) alleles that make up the polymorphism. Two (or more) PCR reactionsare then conducted on sample DNA, each using the common primer and oneof the Allele Specific Primers.

“Splice Variant” as used herein refers to cDNA molecules produced fromRNA molecules initially transcribed from the same genomic DNA sequencebut which have undergone alternative RNA splicing. Alternative RNAsplicing occurs when a primary RNA transcript undergoes splicing,generally for the removal of introns, which results in the production ofmore than one mRNA molecule each of that may encode different amino acidsequences. The term splice variant also refers to the proteins encodedby the above cDNA molecules.

“Identity” reflects a relationship between two or more polypeptidesequences or two or more polynucleotide sequences, determined bycomparing the sequences. In general, identity refers to an exactnucleotide to nucleotide or amino acid to amino acid correspondence ofthe two polynucleotide or two polypeptide sequences, respectively, overthe length of the sequences being compared.

“% Identity”—For sequences where there is not an exact correspondence, a“% identity” may be determined. In general, the two sequences to becompared are aligned to give a maximum correlation between thesequences. This may include inserting “gaps” in either one or bothsequences, to enhance the degree of alignment. A % identity may bedetermined over the whole length of each of the sequences being −23compared (so-called global alignment), that is particularly suitable forsequences of the same or very similar length, or over shorter, definedlengths (so-called local alignment), that is more suitable for sequencesof unequal length.

“Similarity” is a further, more sophisticated measure of therelationship between two polypeptide sequences. In general, “similarity”means a comparison between the amino acids of two polypeptide chains, ona residue by residue basis, taking into account not only exactcorrespondences between a between pairs of residues, one from each ofthe sequences being compared (as for identity) but also, where there isnot an exact correspondence, whether, on an evolutionary basis, oneresidue is a likely substitute for the other. This likelihood has anassociated “score” from which the “% similarity” of the two sequencescan then be determined.

Methods for comparing the identity and similarity of two or moresequences are well known in the art. Thus for instance, programsavailable in the Wisconsin Sequence Analysis Package, version 9.1(Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984, available fromGenetics Computer Group, Madison, Wis., USA), for example the programsBESTFIT and GAP, may be used to determine the % identity between twopolynucleotides and the % identity and the % similarity between twopolypeptide sequences. BESTFIT uses the “local homology” algorithm ofSmith and Waterman (J Mol Biol, 147, 195-197, 1981, Advances in AppliedMathematics, 2, 482-489, 1981) and finds the best single region ofsimilarity between two sequences. BESTFIT is more suited to comparingtwo polynucleotide or two polypeptide sequences that are dissimilar inlength, the program assuming that the shorter sequence represents aportion of the longer. In comparison, GAP aligns two sequences, findinga “maximum similarity” , according to the algorithm of Neddleman andWunsch (J Mol Biol, 48, 443-453, 1970). GAP is more suited to comparingsequences that are approximately the same length and an alignment isexpected over the entire length. Preferably, the parameters “Gap Weight”and “Length Weight” used in each program are 50 and 3, forpolynucleotide sequences and 12 and 4 for polypeptide sequences,respectively. Preferably, % identities and similarities are determinedwhen the two sequences being compared are optimally aligned.

Other programs for determining identity and/or similarity betweensequences are also known in the art, for instance the BLAST family ofprograms (Altschul S F et al, J Mol Biol, 215, 403-410, 1990, Altschul SF et al, Nucleic Acids Res., 25:389-3402, 1997, available from theNational Center for Biotechnology Information (NCBI), Bethesda, Md., USAand accessible through the home page of the NCBI atwww.ncbi.nim.nih.gov) and FASTA (Pearson W R, Methods in Enzymology,183, 63-99, 1990; Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85,2444-2448,1988, available as part of the Wisconsin Sequence AnalysisPackage).

Preferably, the BLOSUM62 amino acid substitution matrix (Henikoff S andHenikoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is usedin polypeptide sequence comparisons including where nucleotide sequencesare first translated into amino acid sequences before comparison.

Preferably, the program BESTFIT is used to determine the % identity of aquery polynucleotide or a polypeptide sequence with respect to areference polynucleotide or a polypeptide sequence, the query and thereference sequence being optimally aligned and the parameters of theprogram set at the default value, as hereinbefore described.

“Identity Index” is a measure of sequence relatedness which may be usedto compare a candidate sequence (polynucleotide or polypeptide) and areference sequence. Thus, for instance, a candidate polynucleotidesequence having, for example, an Identity Index of 0.95 compared to areference polynucleotide sequence is identical to the reference sequenceexcept that the candidate polynucleotide sequence may include on averageup to five differences per each 100 nucleotides of the referencesequence. Such differences are selected from the group consisting of atleast one nucleotide deletion, substitution, including transition andtransversion, or insertion. These differences may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween these terminal positions, interspersed either individually amongthe nucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. In other words, to obtain apolynucleotide sequence having an Identity Index of 0.95 compared to areference polynucleotide sequence, an average of up to 5-25 in every 100of the nucleotides of the in the reference sequence may be deleted,substituted or inserted, or any combination thereof, as hereinbeforedescribed. The same applies mutatis mutandis for other values of theIdentity Index, for instance 0.96, 0.97, 0.98 and 0.99.

Similarly, for a polypeptide, a candidate polypeptide sequence having,for example, an Identity Index of 0.95 compared to a referencepolypeptide sequence is identical to the reference sequence except thatthe polypeptide sequence may include an average of up to fivedifferences per each 100 amino acids of the reference sequence. Suchdifferences are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion. These differences may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between these terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. In other words,to obtain a polypeptide sequence having an Identity Index of 0.95compared to a reference polypeptide sequence, an average of up to 5 inevery 100 of the amino acids in the reference sequence may be deleted,substituted or inserted, or any combination thereof, as hereinbeforedescribed. The same applies mutatis mutandis for other values of theIdentity Index, for instance 0.96, 0.97, 0.98 and 0.99.

The relationship between the number of nucleotide or amino aciddifferences and the

Identity Index may be expressed in the following equation:n _(a) ≦x _(a)−(x _(a) ·I)in which:

-   -   n_(a) is the number of nucleotide or amino acid differences,    -   x_(a) is the total number of nucleotides in SEQ ID NO 1 or SEQ        ID No.3, or amino acids in SEQ ID NO.2 or SEQ ID NO 4,        respectively,    -   I is the Identity Index,    -   · is the symbol for the multiplication operator, and in which        any non-integer product Of x_(a) and I is rounded down to the        nearest integer prior to subtracting it from x_(a).

“Homolog” is a generic term used in the art to indicate a polynucleotideor polypeptide sequence possessing a high degree of sequence relatednessto a reference sequence. Such relatedness may be quantified bydetermining the degree of identity and/or similarity between the twosequences as hereinbefore defined. Falling within this generic term arethe terms “ortholog”, and “paralog”. “Ortholog” refers to apolynucleotide or polypeptide that is the functional equivalent of thepolynucleotide or polypeptide in another species. “Paralog” refers to apolynucleotideor polypeptide that within the same species which isfunctionally similar.

“Fusion protein” refers to a protein encoded by two, unrelated, fusedgenes or fragments thereof. Examples have been disclosed in U.S. Pat.Nos. 5,541,087, 5,726,044. In the case of Fc-PGPCR-3, employing animmunoglobulin Fc region as a part of a fusion protein is advantageousfor performing the functional expression of Fc-PGPCR-3 or fragments ofPGPCR-3, to improve pharmacokinetic properties of such a fusion proteinwhen used for therapy and to generate a dimeric Fc-PGPCR-3. TheFc-PGPCR-3 DNA construct comprises in 5′ to 3′ direction, a secretioncassette, i.e. a signal sequence that triggers export from a mammaliancell, DNA encoding an immunoglobulin Fc region fragment, as a fusionpartner, and a DNA encoding Fc-PGPCR-3 or fragments thereof. In someuses it would be desirable to be able to alter the intrinsic functionalproperties (complement binding, Fc-Receptor binding) by mutating thefunctional Fc sides while leaving the rest of the fusion proteinuntouched or delete the Fc part completely after expression.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

EXAMPLES Example 1 Mammalian Cell Expression

The receptors of the present invention are expressed in either humanembryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. Tomaximize receptor expression, typically all 5′ and 3′ untranslatedregions (UTRs) are removed from the receptor cDNA prior to insertioninto a pCDN or pCDNA3 vector. The cells are transfected with individualreceptor cDNAs by lipofectin and selected in the presence of 400 mg/mlG418. After 3 weeks of selection, individual clones are picked andexpanded for further analysis. HEK293 or CHO cells transfected with thevector alone serve as negative controls. To isolate cell lines stablyexpressing the individual receptors, about 24 clones are typicallyselected and analyzed by Northern blot analysis. Receptor mRNAs aregenerally detectable in about 50% of the G418-resistant clones analyzed.

Example 2 Ligand Bank for Binding and Functional Assays

A bank of over 600 putative receptor ligands has been assembled forscreening. The bank comprises: transmitters, hormones and chemokinesknown to act via a human seven transmembrane (7TM) receptor; naturallyoccurring compounds which may be putative agonists for a human 7TMreceptor, non-mammalian, biologically active peptides for which amammalian counterpart has not yet been identified; and compounds notfound in nature, but which activate 7TM receptors with unknown naturalligands. This bank is used to initially screen the receptor for knownligands, using both functional (i.e. calcium, cAMP, microphysiometer,oocyte electrophysiology, etc, see below) as well as binding assays.

Example 3 Ligand Binding Assays

Ligand binding assays provide a direct method for ascertaining receptorpharmacology and are adaptable to a high throughput format. The purifiedligand for a receptor is radiolabeled to high specific activity (50-2000Ci/mmol) for binding studies. A determination is then made that theprocess of radiolabeling does not diminish the activity of the ligandtowards its receptor. Assay conditions for buffers, ions, pH and othermodulators such as nucleotides are optimized to establish a workablesignal to noise ratio for both membrane and whole cell receptor sources.For these assays, specific receptor binding is defined as totalassociated radioactivity minus the radioactivity measured in thepresence of an excess of unlabeled competing ligand. Where possible,more than one competing ligand is used to define residual nonspecificbinding.

Example 4 Functional Assay in Xenopus Oocytes

Capped RNA transcripts from linearized plasmid templates encoding thereceptor cDNAs of the invention are synthesized in vitro with RNApoiymerases in accordance with standard procedures. In vitro transcriptsare suspended in water at a final concentration of 0.2 mg/ml. Ovarianlobes are removed from adult female toads, Stage V defolliculatedoocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected ina 50 nl bolus using a microinjection apparatus. Two electrode voltageclamps are used to measure the currents from individual Xenopus oocytesin response to agonist exposure. Recordings are made in Ca2+free Barth'smedium at room temperature. The Xenopus system can be used to screenknown ligands and tissue/cell extracts for activating ligands.

Example 5 Microphysiometric Assays

Activation of a wide variety of secondary messenger systems results inextrusion of small amounts of acid from a cell. The acid formed islargely as a result of the increased metabolic activity required to fuelthe intracellular signaling process. The pH changes in the mediasurrounding the cell are very small but are detectable by the CYTOSENSORmicrophysiometer (Molecular Devices Ltd., Menlo Park, Calif.). TheCYTOSENSOR is thus capable of detecting the activation of a receptorwhich is coupled to an energy utilizing intracellular signaling pathwaysuch as the G-protein coupled receptor of the present invention.

Example 6 Extract/Cell Supernatant Screening

A large number of mammalian receptors exist for which there remains, asyet, no cognate activating ligand (agonist). Thus, active ligands forthese receptors may not be included within the ligands banks asidentified to date.

Accordingly, the 7TM receptor of the invention is also functionallyscreened (using calcium, cAMP, microphysiometer, oocyteelectrophysiology, etc., functional screens) against tissue extracts toIdentify natural ligands.

Extracts that produce positive functional responses can be sequenciallysubfractionated until an activating ligand is isolated identified.

Example 7

Calcium and cAMP functional assays 7TM receptors which are expressed inHEK 293 cells have been shown to be coupled functionally to activationof PLC and calcium mobilization and/or cAMP stirnuation or inhibition.Basal calcium levels in the HEK 293 cells in receptor-transfected orvector control cells were observed to be in the normal, 100 nM to 200nM, range. HEK 293 cells expressing recombinant receptors are loadedwith fura 2 and in a single day>150 selected ligands or tissue/cellextracts are evaluated for agonist induced calcium mobilization.Similarly, HEK 293 cells expressing recombinant receptors are evaluatedfor the stimulation or inhibition of cAMP production using standard cAMPquantitation assays. Agonists presenting a calcium transient or cAMPfluctuation are tested in vector control cells to determine if theresponse is unique to the transfected cells expressing receptor.

Example 8 Tissue Expression Primer Design

Primers are designed to orphan GPCR sequences either using thePrimerExpress programme (Applied Biosystems) or manually. Theamplification procedure follows state of the art protocols forquantitative real-time (TaqMan) PCR. Each reaction mixture contains 1×Taqman Universal Master Mix (Applied Biosystems) 0.25 Units PlatiniumTaq DNA polymerase (-Gibco), 50 ng cDNA (Promega), 450 nM each primer,200 nM FAM-labeled TaqMan probe and deionised water in a total volume of25 μl. Cycling is carried out in a 96-well optical reaction plate(Applied Biosystems) using an ABI7700 PCRinstrument. Cycling conditionswere as follows: Pre-activation at 50° C. for 2 min, denaturation at 94°C. for 10 min for 1 cycle then 45 cycles of denaturation at 95° C. for15 s, annealing and extension for 1 min, at 60° C. The kinetics of thereaction is recorded at 488 nm excitation and 518 nm emissionwavelengths. Preparation of cells, RNA and first strand cDNA synthesis

RNA is prepared from cells using SNAP™ extraction kits (Invitrogen)according to the manufacturers protocol. Cells used are primary humanperipheral blood monocytes, monocytes activated by LPS, by IL-10 or acombination of both, human peripheral blood T-cells, lymphocytesactivated by PHA, human peripheral blood B-cells, B-cells activated byanti-CD40 ligation, human moncyte-derived dendritic cells (DC), DCactivated by LPS, IL-10 or a combination of both. Primary humanmonocytes are isolated by elutriation according to standard procedures.T- and B-cells are isolated from peripheral blood according to standardprocedures. Human dendritic cells are prepared from in vitrodifferentiated human peripheral blood monocytes according to standardprotocols.

First strand cDNA is prepared from total RNA isolated from cells usingthe reagents and protocol provided in the first strand cDNA synthesiskit (Roche Molecular Biochemicals).

RT-PCR

Selected sequences are profiled in cDNA derived from tissues and in thedifferent cell types described above by quantitative reversetranscriptase polymerase chain reaction (TaqMan-PCR) followingmanufacturers protocol or semiquantitative PCR. Control reactions areperformed with primers specific for the housekeeping gene EF1α. TissuecDNA's used for RT-PCR profiling are purchased from Clontech. Insemiquantitative PCR, each reaction mixture contains 0.2 mM dNTP's,1×PCR buffer containing 1.5 mM MgCl₂, 0.5 Units Taq DNA polymerase, 50pmol each primer and deionised water in a total volume of 25 μl.Template cDNA used is either from commercial cDNA derived from tissuesamples from Clontech panel I and II (2.5 μl) or from cDNA's preparedfrom cell types as described in the previous section (1 μl). Cycling iscarried out in 0.2 ml tubes using a Biometra Trio PCR machine with theoptimally determined annealing temperature. Cycling conditions are asfollows: Denaturation at 94° C. for 1 min 45 s for 1 cycle then 35cycles of denaturation at 94° C. for 15 s, annealing for 15 s, extensionat 72° C. for 30 s. Reactions are analysed on a 1.5% agarose gel andstained with ethidium bromide. Control RT-PCR reactions are performedwith primers specific to the housekeeping gene GAPDH.

The primer sequences for polynucleotide ORP_(—)9631 are SEQ ID NO.5(forthe forward) and SEQ ID NO 6 (for the reverse) and and SEQ ID NO 7(forthe TaqMan probe). The tissue distribution is given in Table 1.

Sequencing PCR Products

PCR products are excised from agarose gels and purified using theQIAquick spin gel extraction kit (QIAGEN) according to themanufacturer's instructions. PCR products are eluted in 30 μl of sterilewater. Typically 10 ng of purified PCR product is sequenced using one ofthe primers used for amplification and using the BigDye terminator cyclesequencing mix (Applied Biosystems) according to the manufacturer'sinstructions. Sequencing reactions are analysed on an ABI310 sequencer.

Northern Blot Analysis

Northern blot analyses are performed using 12-lane multiple tissuenorthern blots (Clontech) according to the manufacturer's instructions.Briefly, gel purified cDNA fragments (25 ng) were labelled with fresh[α-³²P]dCTP using the Readiprime labelling kit (Amersham) according tothe manufacturer's instructions. Labelled probes are denatured at 95° C.for 5 min then cooled on ice for 5 min immediately prior to use.Prehybridisation is carried out in 5 ml ExpressHyb solution (Clontech)containing 0.1 mg/ml denatured herring sperm DNA at 68° C. for 30 min ina Hybaid oven. Hybridisation is carried out with fresh ExpressHybsolution containing herring sperm DNA (0.1 mg/ml) and denatured probe.Hybridisation is performed at 68° C. for approximately 2 h. Blots arewashed four times with 2×SSC, 0.05% SDS at RT for 10 min followed by 2washes in 0.1×SSC, 0.1% SDS for 20 min at 50° C. Blots are sealed inplastic bags and exposed to a phosphoimager screen (Molecular Dynamics)overnight. Images are processed using a Storm Phosphoimager machine(Molecular Dynamics) and with ImageQuant 5.0 software. Blots arestripped by boiling for 10 min in 0.5% SDS and re-hybridised with aβ-actin (Clontech) ³²P-labelled probe. TABLE 1 Tissue distribution ofCode ORP_9631 PCR Tissue Expression Profiles Score Value brain − 1heart + 22 kidney + 21 liver + 20 lung + 26 pancreas − 2 placenta + 16skeletal muscle + 17 colon − 12 ovary − 7 leukocyte +++ 90 prostate − 4small intestine ++ 73 spleen − 21 testis − 12 thymus − 6 human monocytes+++ 417 activated human monocytes ++ 111 human dendritic cells +++ 783activated human dendritic cells ++ 148 peripheral blood T-cells + 33activated T-cells + 56 peripheral blood B-cells − 6 activated B-cells −5Tissue Expression Score

− not detected

+ weak signal

++ good signal

+++ strong signal

Tissue expression value represents expression level of the test gene Xnormalized to the expression level of the housekeeping gene EF1α (=100000).

Example 9 BLAST Homology Search

The percentage homology at the nucleotide and amino acid level comparedto identified or putative gene ORP_(—)9631 :GENEMBL database:

-   -   BAC clone RP11-378A13 from chromosome 2 (AC021016), 99% identity        FASTA Homology Search in SWISSPROT Database:    -   Mouse gastric histamine H2 receptor (P97292), 28.5% identity in        277 aa overlap (z-score: 256.3)    -   Human gastric histamine H2 receptor (P25021), 26.4% identity in        299 aa overlap (z-score: 243.5)    -   Human sphingosine-1 phosphate receptor EDG8 (Q9H228), 30.3%        identity in 290 aa overlap (z-score: 228.1)    -   Human sphingosine-1 phosphate receptor EDG6 (O95977) 30.5%        identity in 269 aa overlap (z-score: 220.8)    -   Human sphingosine-1 phosphate receptor EDG1 (Q9NYN8) 29.7%        identity in 239 aa overlap (z-score: 216.6)

Example 10 Development of CHO-K1 and HEK Cell Lines Stably ExpressingReceptor ORP_(—)9631

In order to develop stable cell lines expressing the novel receptorORP_(—)9631, plasmid HEDG9631.n31D #5 was linearised using restrictionendonuclease Pvul, which cleaves the plasmid within the ampicillinresistance gene. The linearised plasmid is then precipitated using afinal concentration of 0.3M sodium acetate, pH5.0 and 66% ethanol. Aftercentrifugation and washing using 70% ethanol, the DNA pellet isdissolved in 100 μl water and quantified using a Biosizing 12000 chip(Agilent).

For transfection, CHO-K1 (ATCC Number: CCL-61) and HEK (ATTC;humanembryonic kidney) cells are plated in RPMI/10% FCS medium the day beforetransfection at a cell density of 5×10⁵ cells per well of a 6-wellculture plate (Costar) and incubated at 37° C. in a 5% CO₂ atmosphere.

At the day of transfection, 2 μg of Pvul-linearised HEDG9631.n31 D #5plasmid are added to 100 μl of RPMI medium without fetal calf serum andantibiotics. After adding 10 μl of SuperFect Transfection Reagent(Qiagen, 301305) to the DNA solution and vortexing for 10 seconds, thesample is incubated for 10 min to allow complex formation. TheDNA-SuperFect complex is mixed with an additional 600 μl of RPMI andthen transferred to the cell monolayer, which has been washed once withPBS. After 3 hours of Incubation, the transfection medium is removed andthe cells washed once with PBS. Fresh. RPMI medium supplemented with 10%FCS is added and cells incubated for 2 days at 37° C. and 5% CO₂.Selection for stable transformants Is performed by replacing the mediumwith fresh medium containing 500 μg/ml G418 (Lifetechnologies). Afterabout 12-14 days individual colonies surviving the selection areexpanded and individual cell clones obtained by sorting single cellsinto individual wells of a 96-well cell culture plate using a FACStarPlus cell sorter (BectonDickinson). Individual clones are being expandedand tested for the transcription of ORP_(—)9631 mRNA and G-proteincoupled signal transduction using suitable lipid and small molecularweight compounds.

1. An isolated polypeptide selected from one of the groups consistingof: (a) an isolated polypeptide encoded by a polynucleotide comprisingthe sequence of SEQ ID NO 1 or SEQ ID No.3; (b) an isolated polypeptidecomprising a polypeptide sequence having at least 95% identity to thepolypeptide sequence of SEQ ID NO 2 or SEQ ID NO.4; c) an isolatedpolypeptide having at least 95% identity to the polypeptide sequence ofSEQ ID NO 2 or SEQ ID NO.4; and d) the polypeptide sequence of SEQ ID NO2 or SEQ ID NO.4 and (e)fragments and variants of such polypeptides in(a) to (d).
 2. The isolated polypeptide as claimed in claim 1 comprisingthe polypeptide sequence of SEQ ID NO 2 or SEQ ID NO.4.
 3. An isolatedpolynucleotide selected from one of the groups consisting of: (a) anisolated polynucleotide comprising a polynucleotide sequence having atleast 95% identity to the polynucleotide sequence of SEQ ID NO 1 or SEQID NO.3; (b) an isolated polynucleotide having at least 95% identity tothe polynucleotide of SEQ ID NO 1 or SEQ ID NO 3; (c) an isolatedpolynucleotide comprising a polynucleotide sequence encoding apolypeptide sequence having at least 95% identity to the polypeptidesequence of SEQ ID NO 2 or SEQ ID NO.4; (d) an isolated polynucleotidehaving a polynucleotide sequence encoding a polypeptide sequence havingat least 95% identity to the polypeptide sequence of SEQ ID NO 2 or SEQID NO.4; (e) an isolated polynucleotide with a nucleotide sequence of atleast 100 nucleotides obtained by screening a library under stringenthybridization conditions with a labeled probe having the sequence of SEQID NO 1 or SEQ ID NO 3 or a fragment thereof having at least 15nucleotides; (f) a polynucleotide which is the RNA equivalent of apolynucleotide of (a) to (e); or a polynucleotide sequence complementaryto said isolated polynucleotide and polynucleotides that are variantsand fragments of the above mentioned polynucleotides or that arecomplementary to above mentioned polynucleotides, over the entire lengththereof:
 5. An Isolated polynucleotide as claimed in claim 4 selectedfrom the group consisting of: (a) an isolated polynucleotide comprisingthe polynucleotide of SEQ ID NO 1 or SEQ ID NO 3; (b) the isolatedpolynucleotide of SEQ ID NO 1 or SEQ ID NO 3; (c) an isolatedpolynucleotide comprising a polynucleotide sequence encoding thepolypeptide of SEQ ID NO 2 or SEQ ID NO 4; and (d) an Isolatedpolynucleotide encoding the polypeptide of SEQ ID NO 2 or SEQ ID NO 4.6. An expression system comprising a polynucleotide capable of producinga polypeptide of claim 1 when said expression vector is present in acompatible host cell.
 7. A recombinant host cell comprising theexpression vector of claim 6 or a membrane thereof expressing thepolypeptide of claim
 1. 8. A process for producing a polypeptide ofclaim 1 comprising the step of culturing a host cell as defined in claim7 under conditions sufficient for the production of said polypeptide andrecovering the polypeptide from the culture medium.
 9. A fusion proteinconsisting of the Immunoglobulin Fc-region and any one polypeptide ofclaim
 1. 10. An antibody immunospecific for the polypeptide of any oneof claims 1 to
 3. 11. A method for screening to identify compounds thatstimulate or inhibit the function or level of the polypeptide of claim 1comprising a method selected from the group consisting of: (a) measuringor, detecting, quantitatively or qualitatively, the binding of acandidate compound to the polypeptide (or to the cells or membranesexpressing the polypeptide) or a fusion protein thereof by means of alabel directly or indirectly associated with the candidate compound; (b)measuring the competition of binding of a candidate compound to thepolypeptide (or to the cells or membranes expressing the polypeptide) ora fusion protein thereof in the presence of a labeled competitor; (c)testing whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide, using detection systemsappropriate to the cells or cell membranes expressing the polypeptide;(d) mixing a candidate compound with a solution containing a polypeptideof claim 1, to form a mixture, measuring activity of the polypeptide inthe mixture, and comparing the activity of the mixture to a controlmixture which contains no candidate compound; or (e) detecting theeffect of a candidate compound on the production of mRNA encoding saidpolypeptide or said polypeptide in cells, using for instance, an ELISAassay, and (f) producing said compound according to biotechnological orchemical standard techniques.
 12. A process for diagnosing a disease ora susceptibility to a disease related to the expression or activity ofthe polypeptides SEQ ID No.2 or SEQ ID NO.4 comprising: (a) determiningthe presence or absence of mutation in the nucleotide sequence encodingsaid Polypeptide in the genome of said subject: and/or (b) analysing forthe present or amount of the polypeptide expression in a sample derivedfrom said subject.